The subject of the present invention is a device for the actively controlled, localized deposition of at least one biological solution in the form of microdrops.
In the pharmaceutical industry, the capital invested in research for developing new medicines occupies a considerable portion of an enterprise's budget.
New assay methods are needed to reduce the cost of such research.
The arrival of microchips in the biomedical sector has revolutionized the fields of medicinal product development and of bioassay.
The advantages of these microchips are the following:
they allow new, more sensitive detection methods to be developed;                they require smaller volumes of reactants, hence a lower cost;        they allow analytical procedures that are more rapid, owing to their small dimensions; and        they allow screening or diagnostic studies to be carried out owing to the large number of different solutions present on any one surface.        
However, the tools that are currently operational for distributing small volumes of biological material in solution allow the deposition, on glass slides or on membranes, of drops with a diameter of the order of a hundred microns (which corresponds to a drop volume of the order of a nanoliter). These systems rely:                either, in a first case, on a piezoelectric active device for sucking up and ejecting the products in solution (a contactless deposition system);        or, in a second case, on a passive mechanism consisting of split pins made of metal (stainless steel, tungsten, etc.), the liquid being sucked up in this second case by capillary effect and being deposited by bringing the end of the pin into contact with a glass slide (a contact deposition system). We should also mention the pin-and-ring system, the operating principle of which is similar to that used with the mechanism consisting of split pins, the ring acting as a liquid reservoir in this case.        
Other deposition techniques that have formed the subject of laboratory studies are known, these making it possible to achieve smaller volumes than those obtained with the abovementioned operational tools.
One of these techniques is dip-pen lithography, which is a technique derived from atomic force microscopy and makes it possible to form features on a surface using a molecular transport diffusion effect at the water meniscus that forms between the tip of an atomic force microscope and the surface on which the deposition takes place. The operating principle relies on the difference in hydrophilicity or wettability properties between the tip and the surface. The surface must in fact be more hydrophilic than the tip in order to cause molecular diffusion from the tip toward the surface. The resolution obtained may be less than 1 micron and it is also possible to envisage the deposition of different biological molecules, but this means changing the tip (which will have been immersed beforehand in the solution to be deposited) for each solution. This deposition technique is therefore extremely time consuming if it is desired to carry out several tens of different depositions. Moreover, changing the tip of the microscope does not make it possible to maintain the alignment precision between two changes. Finally, this approach can be implemented only under high humidity conditions in order for the water meniscus to form.
This technique is described in particular in the following articles:                “Dip-pen Nanolithography” R. D. Piner, J. Zhu, F. Xu, S. Hong and C. A. Mirkin, Science, Vol. 283, pages 661-663, Jan. 29, 1999;        “Multiple Ink Nanolithography : toward a Multiple-Pen Nano-Plotter”, S. Hong, J. Zhu and C. A. Mirkin, Science, Vol. 286, pages 523-525, Oct. 15, 1999;        “Surface organization and nanopatterning of collagen by dip-pen nanolithography”, D. L. Wilson, R. Martin, S. Hong, M. Cronin-Golomb, C. A. Mirkin and D. L. Kaplan, Proceedings of the National Academy of Sciences of the United States of America, Volume 98, Issue 24, Nov. 20, 2001, pages 13660-13664; and        “Dip-Pen nanolithography on semiconductor surfaces”, A. Ivanisevic and C. A. Mirkin, Journal of the American Chemical Society, Volume 123, Issue 32, Aug. 15, 2001, pages 7887-7889.        
Other Microsystems have also been proposed for carrying out depositions for the fabrication of biochips. These apply in general to microfluid structures, for example that described in the following article:                “Micromachined needle arrays for drug delivery or fluid extraction”, IEEE Engineering in Medicine and Biology Magazine: the Quarterly Magazine of the Engineering in Medicine & Biology Society, Volume 18, Issue 6, November-December 1999, pages 53-58, J. Brazzle, I. Papautsky and A. B. Frazier.        
These are micromachined silicon structures having microfabricated channels, and their use is altogether comparable to that of an ink jet system. These “closed” structures, in the form of tubes, are very difficult to clean, which represents an obstacle to the same device being used to deposit droplets of different liquids.
International patent application WO 02/00348 illustrates a deposition system that allows microdroplets with a volume of between 10 picoliters and 200 nanoliters to be deposited. Such a system consists of at least one lever, made of silica or quartz, equipped with a capillary channel and with a reservoir. The liquid is picked up and deposited purely passively, by capillary effect and by the difference in wettability between the device and the deposition surface.
Micropipettes allowing contactless deposition, by means of a field effect, are described in particular in the following documents:                “Electrospray deposition as a method for a mass fabrication of mono and multicomponent microarrays of biological and biologically active substances”, V. N. Morozov and T. Ya. Morozova, Analytical Chemistry, Volume 71, Issue 15, Aug. 1, 1999, pages 3110-3117; and        “Atomic force microscopy of structures produced by electrospraying polymer solutions”, Victor N. Morozov, Tamara Ya Morozova and Neville R. Kallenbach, International Journal of Mass Spectrometry, Volume 178, Issue 3, Nov. 9, 1998, pages 143-159.        
These devices exploit the electrospray effect in order to deposit in a controlled manner, by means of an adjustable electric field, very small amounts of organic molecules. However, the electrospray method consists in applying an electric field high enough to ionize and atomize the liquid to be deposited. The droplets thus produced have submicron dimensions and evaporate before they reach the deposition surface; in this way, thin films are produced. This is therefore a different problem from that facing the present invention, that is to say the deposition of droplets with a volume of the order of 1 picoliter or 1 femtoliter. In addition, the electrospray devices consist of micropipettes containing a needle-shaped electrode; they cannot therefore be effectively washed and have to be replaced each time the liquid is changed.
Studies on surface wetting under the effect of an electric field and the displacement of a liquid by actively controlling the wettability of a surface have been published in the following articles:                “Electrowetting and electrowetting-on-dielectric for microscale liquid handling”, J. Lee, H. Moon, J. Fowler, T. Schoellhammer and C. J. Kim, Sensors and Actuators, A 95, pages 259-268, 2002; and        “Dielectrophoretic liquid actuation and nanodroplet formation”, T. B. Jones, M. Gunji, M. Washizu and M. J. Feldman, Journal of Applied Physics, Vol. 89, No. 2, pages 1441-1448, 2001.        
These articles describe the physical principles of electrowetting and dielectrophoresis, and also their application for handling droplets of liquids such as water. Although these effects have been known for several decades, they have never been applied to the deposition of liquid droplets.
In conclusion, no deposition system has yet been proposed that allows microdrops with a diameter of less than 10 microns, that is to say with a volume of less than 1 picoliter (p1), to be deposited in an actively controlled and precise (relative to a reference) manner.
A fortiori, no known deposition system allows such drops to be deposited in a precise and actively controlled manner on microstructures of the bridge, beam or membrane type.